[EC 1. 6. 99. -]
from Bacillus stearothermophilus
|Specific activity||:||more than 1,000 U/mg protein|
|Contaminants||:||(as Diaphorase activity = 100%)|
|Molecular weight||:||ca. 30,000|
|pH stability||:||7.5 - 9.5||(Fig.2)|
|Thermal stability||:||No detectable decrease in activity
up to 50°C.
|Michaelis constants||:||See Table 1|
|Substrate specificity||:||See Table 1|
|Effectors||:||cations and anions||(Fig.5,6)|
stable at -20 to 5°C for at least one year
The enzyme is useful for the measurement of various dehydrogenase reactions in visible spectral range.
The change in absorbance is measured at 600 nm according to the following reaction.
One unit of activity is defined as the amount of Di-1 that reduces 1 μmol of DCIP per minute at 30°C.
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 1.0 to 2.0 U/mL with 50mM potassium phosphate buffer, pH 7.5.
|Solution II||2.28 mL|
|Table 1. SUBSTRATE SPECIFICITY OF DIAPHORASE I|
|Km Acceptor (mM)||0.015||0.15||0.40||-|
|Km NADH (mM)||0.50||0.02||0.07||-|
|Km NADPH (mM)||0.52||0.19||0.50||-|
|Activity NADH (U/mg)||1,200||225||290||18|
|Activity NADPH (U/mg)||4||150||120||-|
|(pH 8.5)||50mM||(pH 7)||50mM||(pH 7.5)||96mM||(pH 7)||88mM|
|Triton X-100||0.1%||(2 %-DMSO)|
Effect of BSA on the activity of DIAPHORASE I:
BSA stimulates the activity with INT as electron acceptor and the activation can be increased 30 fold with concentrations above 1 mg/mL BSA (Fig. 10). The extent of activation for DCIP is about 35 %, whereas the activities with NBT and FMN are not affected by BSA.
Effect of Triton X-100 on the activity of DIAPHORASE I:
The activity with NBT is little in the absence of Triton X-100, but is greatly increased by the addition of Triton X-100 (Fig. 8). On the other hand, Triton X-100 has no effect on the activities with DCIP, INT and FMN.